(1) Field of the Invention
The present invention generally relates to enzyme products and activities. More particularly, the invention is directed to the discovery of products and activities of Sir2 enzymes.
(2) Description of the Related Art
References Cited
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The Sir2p-like enzymes are broadly conserved from bacteria to humans1,2. In yeast, these proteins form complexes with other proteins to silence chromatin3-6 by accessing histones7,8 and deacetylating them9-12. Sir2 enzymes are homologs of the bacterial enzyme cobB, a phosphoribosyltransferase13, which led to the finding that Sir2p employs NAD+ as a co-substrate in deacetylation reactions10,14,12. This unusual requirement for NAD+ is stoichiometric14 and generates a novel product originally proposed to be β-1′-AADPR15,16 or possibly 2′-AADPR16,17. A crystal structure of a Sir2p homolog from Archaeoglobus fulgidus called Sir2-Af1 was recently determined with NAD+ bound at the active site. The structure was interpreted in the context of a catalytic mechanism that produces β-1′-AADPR18. A cleft is proposed to bind the acetyl-lysine side chain of substrate proteins in proximity to the C1′ of the bound NAD+, providing substrate organization for acetyl group transfer between the peptide side chain and NAD+18.
From mechanistic and thermodynamic considerations, the NAD+ dependent deacetylation by Sir2p is an unusual reaction, since Lys N-deacetylation reactions are simple to accomplish by hydrolysis alone. The apparent coupling of hydrolysis and ADP-ribose transfer to acetate forms acetyl-ADP-ribose (AADPR) as product, and generates a new metabolite of unknown function15-17.
Evidence to date indicates that histones are in vivo substrates of Sir2 enzymes. Yet, the genomes of eubacteria such as Salmonella which lack histones, encode Sir2-like proteins19. Similarly, archaeal genomes that encode Sir2ps also encode histone-like proteins, but those histones lack the N-terminal tails that are the prominent sites of lysine acetylation in eukaryotic histones. One archaeal species, Archaeoglobus fulgidus, encodes two different Sir2-like deacetylases that are 47% identical and the X-ray structure of one of these (Sir2Af1) was recently determined18. Sir2Af2 has been characterized more extensively biochemically, and is active in vitro on defined substrates12.
Histone deacetylation by Sir2p is proposed to be the major reaction that converts chromatin from active to silent states9,10,12. This suggestion is supported by studies of silencing complexes which show Sir2p protein is the only conserved SIR family member in all yeast silencing complexes studied28. Caloric restriction upregulates Sir2p activity and may extend lifespan, showing that silencing might have potent biological effects in various organisms29,30. The requirement for NAD+ and evidence that silencing factors can “sense” the redox state of the cell suggests that Sir2p family members are unique amide-hydrolysis enzymes with broad roles12,31.
The distribution of Sir2p family of enzymes into organisms without histone substrates, and eukaryotic genomes encoding multiple Sir2 proteins, suggest a family of deacetylases with varying substrates. Mutagenesis experiments suggest that the N- and C-terminal regions flanking the catalytic core domain of Sir2p help direct it to different targets32. Although most Sir2 proteins in eukaryotic cells are located in the nucleus33 others are cytosolic34-36, or even mitochondrial (Onyango, Celic, Boeke and Feinberg; unpublished observations) which suggests additional substrates.